Streptavidin peroxidase conjugate

A standard biotin-streptavidin-peroxidase method was used for all immunohistochemical staining. Reagents used in this study were monoclonal antibodies against CD68 (macrophages, 1:200, clone #: FA-11, Cat#: 137002), CD4 (CD4⁺ T cells, 1:200, clone #: GK1.5, Cat#: 100402), CD8 (CD8⁺ T cells, 1:200, clone #: 53-6.7, Cat#: 100702), B220 (B cells, 1:200, clone #: RA3-6B2, Cat#: 103202) and CD31 (1:200, clone#: 390, Cat#: 102402) (all above mentioned primary antibodies from Biolegend Inc, San Diego, CA, USA), goat anti-mouse polyclonal antibodies against MMP2 (1:200, Cat#: AF1488, R&D Systems) and MMP9 (1:200, Cat#: AF909, R&D Systems), a biotinylated goat anti-rat secondary antibody (1:400, Cat#: BA-9400, Vector Laboratories, Inc) or rabbit anti-goat IgG secondary antibody (1:400, Cat#: BA-5000, Vector Laboratories, Inc), and streptavidin-peroxidase conjugate (1:200, Cat#: 016-030-084, Jackson ImmunoResearch) (27 (link)–32 (link)). Macrophage accumulation was scored as the grade I to IV, and the densities of CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells and angiogenesis were quantified as the number of positively stained cells or neovessels per aortic cross section (ACS) (30 (link)). MMP expression levels were quantified as a positively stained area percentage of aortic wall using WinRood 6.5 image software, Mitani Co. Ltd., Tokyo, Japan.

Acetone-fixed frozen aortic sections were prepared from mice 14 days after PPE infusion and used for Verhoeff’s Van Gieson (EVG) stain and immunohistochemistry as described previously [29 (link)]. Monoclonal antibodies (mAb) for immunohistochemistry were biotinylated anti-SMC α actin (Clone 1A4 from Thermo Fisher Scientific Inc, Waltham, MA, USA as well as CD68 (Clone FA-11 for macrophages), CD4 (Clone GK 1.5), CD8 (Clone 53-5.8), B220 (Clone RA3-6B2 for B cells) and CD31 (Clone 390 for blood vessels) from Biolegend Inc, San Diego, CA, USA. Biotinylated anti-rat IgG (Catalog number 112-065-006) and streptavidin-peroxidase conjugate (Catalog number 016-030-084) were obtained from Jackson ImmunoResearch Inc., West Grove, PA, USA. Peroxidase substrate AEC (3-amino-9-ethylcarbazole) kits were purchased from Vector Laboratories, Burlingame, CA, USA. Aortic macrophage accumulation, medial elastin degradation and SMC loss were scored as grade I (mild) to IV (severe) using previously reported histological grading criteria [29 (link)]. Other subsets of leukocytes and neovessels were quantitated as the number of leukocyte subset-antibody positive cells and CD31-positive vessels per ACS, respectively [29 (link)]. All histological assessments were analyzed by a single experienced experimental pathologist who was blinded to experimental group assignment.

Permeabilized muscle fibres were treated similarly to those described for the identification of protein SH groups. Briefly, preparations were exposed to 2 μM biotin-1-3-cyclopentanedione (BP1) (Kerafast, Boston, MA, USA) in 50 mM Bis-Tris citric acid buffer (pH 5.5) [47 (link)]. After 90 min of labeling with BP1, muscle fibres were solubilized in urea buffer for 45 min. Then 10 µg protein homogenates were loaded on 2.1 and 4% polyacrylamide gels and after SDS-PAGE the separated proteins were transferred onto nitrocellulose membrane. Sypro Ruby Protein Blot Stain (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used to determine the protein amounts. Free protein binding sites on the membranes were blocked by milk powder (10%) for one hour and then streptavidin-peroxidase conjugate (Jackson ImmunoResearch, West Grove, PA, USA) was used (60 min incubation) to identify the biotinylated proteins. SOH groups were identified by ECL method and normalized for those assessed with Sypro Rubi Protein Blot Stain.

Mice were euthanized by carbon dioxide inhalation 14 days following PPE infusion. Aortas were harvested, embedded in optical cutting temperature compound, sectioned (6 μm) and fixed with cold acetone. Infrarenal aortas from naïve WT mice were processed identically and served as non-aneurysmal controls. Frozen sections were stained with hematoxylin and eosin (H&E) and a standard streptavidin peroxidase immunohistochemical method for the assessment of morphological alterations and CRP protein expression, respectively. Reagents for CRP tissue immunostaining were a rabbit anti-mouse CRP polyclonal antibody (1:200, Cat#: bs-0155R) and normal rabbit IgG (1:200, Cat#: bs-0295P) from Bioss Technology. Other reagents were biotinylated goat anti-rabbit IgG antibody (1:400, Cat#: BA-1000) and AEC substrate kit (Cat#: SK-4200, Vector Laboratories, Inc, Newark, CA, USA) and streptavidin-peroxidase conjugate (1:200, Cat#: 016-030-084, Jackson ImmunoResearch, West Grove, PA, USA).